CN110734475B - 一种具有α-葡萄糖苷酶抑制活性的寡肽及其应用 - Google Patents
一种具有α-葡萄糖苷酶抑制活性的寡肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有α‑葡萄糖苷酶抑制活性的寡肽,寡肽的缩写为AVPANLVDLNVPALLK,分子量1647.8Da,所述寡肽的氨基酸序列为Ala‑Val‑Pro‑Ala‑Asn‑Leu‑Val‑Asp‑Leu‑Asn‑Val‑Pro‑Ala‑Leu‑Leu‑Lys,所述寡肽可以从黑茶千两茶中分离得到,也可以使用固相合成仪合成得到,能够实现体外降血糖的效果,可应用于保健食品、特殊医学用途配方食品与生物制药领域,改善糖尿病病人的病症,本发明还公布了所述寡肽的制备方法、含有该寡肽的药品组合物、含有该寡肽的保健品和含有该寡肽的食品。
Description
技术领域
本发明涉及生物制药领域,特别是一种具有α-葡萄糖苷酶抑制活性的寡肽,本发明还涉及该寡肽的制备方法和应用。
背景技术
糖尿病的早期征兆往往是餐后高血糖,特别是2型糖尿病病人,,餐后高血糖不仅极易诱发各种并发症,还会极大地提高糖尿病的死亡率,而α-葡萄糖苷酶位于小肠刷状缘膜上皮细胞,它能将双糖,如蔗糖、麦芽糖等水解成可被小肠吸收的单糖,从而导致血糖升高,因此,抑制α-葡萄糖苷酶能够延缓碳水化合物的消化,减少葡萄糖吸收入血,进而控制餐后血糖浓度的升高。
α-葡萄糖苷酶抑制剂是一类新型的降血糖药物,它可通过抑制小肠刷状缘细胞表面的α-葡萄糖苷酶活性,减缓葡萄糖的生成及吸收,同时增加肝脏及周围组织对胰岛素的敏感性,减少高血糖对胰腺刺激,从而起到降低血糖浓度糖及治疗糖尿病的目的。虽然,合成的α-葡萄糖苷酶抑制药物已经上市(如阿卡波糖等),但由于它们有一定的毒副作用,因此从植物中筛选天然的α-葡萄糖苷酶抑制剂具有广阔的前景。
发明内容
本发明旨在提供一种具有α-葡萄糖苷酶抑制活性的寡肽及其应用。
本发明解决其技术问题所采用的技术方案是:
一种具有α-葡萄糖苷酶抑制活性的寡肽,所述寡肽的氨基酸序列为Ala-Val-Pro-Ala-Asn-Leu-Val-Asp-Leu-Asn-Val-Pro-Ala-Leu-Leu-Lys,所述寡肽的缩写为AVPANLVDLNVPALLK,分子量1647.8Da。
所述寡肽可以从黑茶千两茶中分离得到。具体包括如下步骤:
(1)茶蛋白提取:将千两茶叶粉碎后过20-40目筛,以料液比为1:5-40的比例混合茶粉和超纯水,于95℃加热浸提10-60min,浸提液过滤后在25℃,8000rpm/min的条件下离心20min,收集上清液,往上清液中加入饱和硫酸铵溶液(上清液:硫酸铵=1:2-6),在4℃静置12h,使蛋白质沉淀,然后,于4℃,6000rpm/min条件下离心20min,取沉淀,用超纯水溶解,在室温中透析24小时,以去除硫酸铵等小分子物质,获得千两茶粗蛋白;
(2)粗蛋白的酶解:将步骤(1)制得的千两茶粗蛋白样品制成2-10mg/mL的溶液,于50℃水浴中保持10min,用浓度为2.0mol/L的氢氧化钠调节PH至8.0,以酶/底物比(E/S)为1-5%(w/w)的比例添加胰蛋白酶,酶解时间为5-10h,酶解过程中用浓度为2.0mol/L的氢氧化钠溶液调节酸碱度,使其保持在pH 8.0,酶解完成后把样品置于95℃下加热20min使酶灭活,并于4℃,8000rpm/min离心10min,弃去沉淀,制得千两茶胰蛋白酶酶解组分,冻干备用;
(3)“酶-抑制肽”复合物的制备:取所述千两茶胰蛋白酶酶解组分配成1-5mg/mL样品溶液,将α-葡萄糖苷酶溶解在10mM pH 6.8磷酸盐缓冲液中配成10U/mL的α-葡萄糖苷酶溶液,再取50-300μL所述样品溶液与100-500μL所述α-葡萄糖苷酶溶液混合,于37℃下,摇床培养10-60min,使酶与抑制剂样品充分结合;
(4)未与酶结合成分的去除:培养结束后,把步骤(3)制得的混合物加入截留分子量为30kDa的超滤离心管中,并于常温下,10000rpm/min离心25min,去除样品中未与酶结合的成分,用200μL磷酸缓冲洗截留后的滤液3次,并于常温下,5000rpm/min离心5min;
(5)抑制肽的释放、分离与鉴定:离心后往上层(即“酶-抑制肽”复合物层)液体中加入100-400μL 50%乙腈水溶液,混合均匀,静置10min之后,在常温下以10000rpm/min离心10分钟,重复2次,合并滤出液,过0.22μm超滤膜,制得千两茶超滤液,进行高效液相谱分离,收集合适的峰,即获得所述具有α-葡萄糖苷酶抑制活性的寡肽。
所述寡肽也可以使用固相合成仪合成制得,也就是说,将目标多肽的C-端羧基以共价键形式与一个不溶性的高分子树脂相连,然后以这个氨基酸的氨基作为起点,与另一分子氨基酸的羧基作用形成肽键,不断重复这一过程,合成反应完成后,去除保护基,将肽链与树脂分离,即得到目标产物。多肽合成是一个重复添加氨基酸的过程,固相合成顺序从C端向N端合成。
一种药物组合物,所述药物组合物含有上述的具有α-葡萄糖苷酶抑制活性的寡肽。
一种保健品,所述保健品含有上述的具有α-葡萄糖苷酶抑制活性的寡肽。
一种食品,所述食品含有上述的具有α-葡萄糖苷酶抑制活性的寡肽。
所述寡肽在浓度为2mg/mL时,对α-葡萄糖苷酶的体外抑制率为94.3%。
所述寡肽在浓度为1.5mg/mL时,对α-葡萄糖苷酶的体外抑制率为84.6%。
所述寡肽对α-葡萄糖苷酶的50%抑制浓度(IC50)为1.0261±0.12mg/mL。
本发明的有益效果是:可以从黑茶千两茶提取出一种具有α-葡萄糖苷酶抑制活性的寡肽,能够实现体外降血糖的效果,可应用于保健食品、特殊医学用途配方食品与生物制药领域,改善糖尿病病人的病症。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是实施例1所述千两茶超滤液的高效液相色谱图;
图2是实施例1所述寡肽的一级质谱图;
图3是实施例1所述寡肽的二级质谱图;
图4是实施例1所述寡肽的离子碎片分析图;
图5是实施例2所述寡肽的高效液相色谱图;
图6是实施例2所述寡肽的质谱图;
图7是本发明所述寡肽与阿卡波糖的“浓度-抑制率”折线图。
具体实施方式
实施例1:
一种制备具有α-葡萄糖苷酶抑制活性的寡肽的方法,包括如下步骤:
(1)茶蛋白提取:将千两茶叶粉碎后过20目筛,以料液比为1:20的比例混合茶粉和超纯水,于95℃加热浸提30min,浸提液过滤后在25℃,8000rpm/min的条件下离心20min,收集上清液,往上清液中加入饱和硫酸铵溶液(上清液:硫酸铵=1:4),在4℃静置12h,使蛋白质沉淀,然后,于4℃,6000rpm/min条件下离心20min,取沉淀,用超纯水溶解,在室温中透析24小时,以去除硫酸铵等小分子物质,获得千两茶粗蛋白;
(2)粗蛋白的酶解:将步骤(1)制得的千两茶粗蛋白样品制成5mg/mL的溶液,于50℃水浴中保持10min,用浓度为2.0mol/L的氢氧化钠调节PH至8.0,以酶/底物比(E/S)为1.5%(w/w)的比例添加胰蛋白酶,酶解时间为6h,酶解过程中用浓度为2.0mol/L的氢氧化钠溶液调节酸碱度,使其保持在pH 8.0,酶解完成后把样品置于95℃下加热20min使酶灭活,并于4℃,8000rpm/min离心10min,弃去沉淀,制得千两茶胰蛋白酶酶解组分,冻干备用;
(3)“酶-抑制肽”复合物的制备:取千两茶胰蛋白酶酶解组分配成2mg/mL样品溶液,将α-葡萄糖苷酶溶解在10mM pH 6.8磷酸盐缓冲液中配成10U/mL的α-葡萄糖苷酶溶液,再取100μL所述样品溶液与200μL所述α-葡萄糖苷酶溶液混合,于37℃下,摇床培养30min,使酶与抑制剂样品充分结合;
(4)未与酶结合成分的去除:培养结束后,把步骤(3)制得的混合物加入截留分子量为30kDa的超滤离心管中,并于常温下,10000rpm/min离心25min,去除样品中未与酶结合的成分,用200μL磷酸缓冲洗截留后的滤液3次,并于常温下,5000rpm/min离心5min;
(5)抑制肽的释放、分离与鉴定:离心后往上层(即“酶-抑制肽”复合物层)液体中加入200μL 50%乙腈水溶液,混合均匀,静置10min之后,在常温下以10000rpm/min离心10分钟,重复2次,合并滤出液,过0.22μm超滤膜,制得千两茶超滤液,进行高效液相谱分离。
所用的高效液相色谱仪为Angilent 1100,相关参数设置:A相为水,B相为乙腈,0-2min为85%水,15%乙腈;6-9.5min为15%水,85%乙腈;10-12min为85%水,15%乙腈,流速为0.2mL/min。高效液相色谱分离结果如图1所示,收集第7个峰即获得具有α-葡萄糖苷酶抑制活性的寡肽,分子量1647.8Da,所述寡肽的氨基酸序列为Ala-Val-Pro-Ala-Asn-Leu-Val-Asp-Leu-Asn-Val-Pro-Ala-Leu-Leu-Lys,所述寡肽的缩写为AVPANLVDLNVPALLK。
质谱参数设置:ESI离子源,扫描范围50m/z-3000m/z,阳离子模式,端板偏移-500V,电压变化2000V,正离子谱测定,分子量测定范围在500-3000KD。质谱鉴定如图2至图3所示。
实施例2:
采用固相合成仪合成所述具有α-葡萄糖苷酶抑制活性的寡肽,具体操作为:
(1)通过树脂的筛选合成所述寡肽,选用高分子树脂(王氏树脂,上海阿拉丁生化科技股份有限公司),按照氨基酸序列Ala-Val-Pro-Ala-Asn-Leu-Val-Asp-Leu-Asn-Val-Pro-Ala-Leu-Leu-Lys的特征,先将Ala的羧基以共价键的形式与一个树脂的连接位点相连,然后Ala的氨基和Val的羧基缩水反应,处理后,再添加Pro,Pro的氨基和另一个Ala的羧基反应,依次从右到左添加氨基酸,加好最后一个Lys氨基酸后,再切除树脂,即得到目标多肽;
(2)采用高效液相色谱进行纯化,色谱柱型号为Phenomenex C18,尺寸4.6*150mm,流动相A:含有0.1%三氟乙酸(TFA)的乙腈;流动相B:含有0.1%TFA的水;25min内B相由95.0%下降到30.0%,流速1.0mL/min,检测波长214nm;
(3)液氮速冻,冷冻干燥,即制得所述具有α-葡萄糖苷酶抑制活性的寡肽,该成品纯度为96.89%,分子质量为1647.8Da,高效液相色谱分离结果如图5所示,质谱鉴定如图6所示。
对α-葡萄糖苷酶的体外抑制活性实验:
选取实施例2制得的寡肽分别配成浓度为0.125mg/mL、0.25mg/mL、0.5mg/mL、1mg/mL、1.5mg/mL和2mg/mL的寡肽溶液,以α-葡萄糖苷酶为研究对象,测定寡肽的体外抑制活性。
在黑色96孔板中均加入10μLα-葡萄糖苷酶的酶溶液(浓度0.2U/mL)和50μL 0.2M的磷酸缓冲液(pH 6.8),再分别加入各个浓度的20μL寡肽溶液,混合均匀后于37℃摇床反应20min,后加入40μL底物溶液(5mM p-NPG溶液),于37℃摇床反应30min,最后加入140μLNa2CO3溶液,使反应终止,于405nm处测吸光值。
对照组加入缓冲液20μL和α-葡萄糖苷酶的酶溶液10μL,背景组加入样品溶液20μL和缓冲液10μL,阳性对照组分别为20μL相同浓度(即0.125mg/mL、0.25mg/mL、0.5mg/mL、1mg/mL、1.5mg/mL、2mg/mL和4mg/mL)的阿卡波糖溶液和α-葡萄糖苷酶的酶溶液10μL,其余操作与上述相同。
α-葡萄糖苷酶的抑制率的计算公式如下:
其中,Aa是对照组的吸光值,A0是背景组的吸光值,Ab是实验组的吸光值。
对每个孔的数据作“浓度-抑制率”图的线性图象,如图7所示,由图可知所述寡肽对α-葡萄糖苷酶的50%抑制浓度(IC50)为1.0261±0.12mg/mL;本发明的寡肽对α-葡萄糖苷酶的抑制率低于阿卡波糖,但是对于糖尿病的治疗仍有指导意义,可应用于保健食品、特殊医学用途配方食品与生物制药领域,改善糖尿病病人的病症。
实施例3:
一种药物组合物,含有α-葡萄糖苷酶抑制活性的寡肽和药学上可接受的载体或赋形剂,所述寡肽可以从黑茶千两茶中分离得到,所述寡肽的缩写为AVPANLVDLNVPALLK,分子量1647.8Da,氨基酸序列为Ala-Val-Pro-Ala-Asn-Leu-Val-Asp-Leu-Asn-Val-Pro-Ala-Leu-Leu-Lys。本实施例为注射剂,主要成分为所述寡肽,其他成分均为制药领域注射剂的常规辅料。
实施例4:
一种保健品,含有α-葡萄糖苷酶抑制活性的寡肽,所述寡肽的缩写为AVPANLVDLNVPALLK,分子量1647.8Da,氨基酸序列为Ala-Val-Pro-Ala-Asn-Leu-Val-Asp-Leu-Asn-Val-Pro-Ala-Leu-Leu-Lys,所述寡肽可以从黑茶千两茶中分离得到,其他成分均为保健品领域的常规添加材料。
实施例5:
一种食品,含有具有α-葡萄糖苷酶抑制活性的寡肽,所述寡肽的缩写为AVPANLVDLNVPALLK,分子量1647.8Da,氨基酸序列为Ala-Val-Pro-Ala-Asn-Leu-Val-Asp-Leu-Asn-Val-Pro-Ala-Leu-Leu-Lys,所述寡肽可以从黑茶千两茶中分离得到,其他成分均是根据食品类型进行添加制作。
以上的实施方式不能限定本发明创造的保护范围,专业技术领域的人员在不脱离本发明创造整体构思的情况下,所做的均等修饰与变化,均仍属于本发明创造涵盖的范围之内。
Claims (6)
1.一种具有α-葡萄糖苷酶抑制活性的寡肽,其特征在于所述寡肽的缩写为AVPANLVDLNVPALLK,所述寡肽的氨基酸序列为Ala-Val-Pro-Ala-Asn-Leu-Val-Asp-Leu-Asn-Val-Pro-Ala-Leu-Leu-Lys;
所述寡肽可以从黑茶千两茶中分离得到;
所述寡肽的制备步骤如下:
(1)茶蛋白提取:从千两茶叶中提取千两茶粗蛋白;
(2)粗蛋白的酶解:将步骤(1)制得的千两茶粗蛋白酶解,制得千两茶胰蛋白酶酶解组分,冻干备用;
(3)“酶-抑制肽”复合物的制备:取所述千两茶胰蛋白酶酶解组分配成1-5mg/mL样品溶液,将α-葡萄糖苷酶溶解在10mM pH 6.8磷酸盐缓冲液中配成10U/mL的α-葡萄糖苷酶溶液,再取50-300μL所述样品溶液与100-500μL所述α-葡萄糖苷酶溶液混合,于37℃下,摇床培养10-60min,使酶与抑制剂样品充分结合;
(4)未与酶结合成分的去除:培养结束后,把步骤(3)制得的混合物加入截留分子量为30kDa的超滤离心管中,并于常温下,10000rpm/min离心25min,去除样品中未与酶结合的成分,用200μL磷酸缓冲洗截留后的滤液3次,并于常温下,5000rpm/min离心5min;
(5)抑制肽的释放、分离与鉴定:离心后往上层的“酶-抑制肽”复合物层液体中加入100-400μL 50%乙腈水溶液,混合均匀,静置10min之后,在常温下以10000rpm/min离心10分钟,重复2次,合并滤出液,过0.22μm超滤膜,制得千两茶超滤液,进行高效液相谱分离,收集合适的峰,即获得所述具有α-葡萄糖苷酶抑制活性的寡肽。
2.一种药物组合物,其特征在于所述药物组合物含有权利要求1所述的具有α-葡萄糖苷酶抑制活性的寡肽。
3.根据权利要求1所述的具有α-葡萄糖苷酶抑制活性的寡肽,其特征在于所述寡肽可以使用固相合成仪合成制得。
4.根据权利要求1所述的具有α-葡萄糖苷酶抑制活性的寡肽,其特征在于所述寡肽在浓度为2mg/mL时,对α-葡萄糖苷酶的体外抑制率为94.3%。
5.根据权利要求1所述的具有α-葡萄糖苷酶抑制活性的寡肽,其特征在于所述寡肽在浓度为1.5mg/mL时,对α-葡萄糖苷酶的体外抑制率为84.6%。
6.根据权利要求1所述的具有α-葡萄糖苷酶抑制活性的寡肽,其特征在于所述寡肽对α-葡萄糖苷酶的50%抑制浓度(IC50)为1.0261±0.12mg/mL。
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Keying Su et al.,.In vitro assessment of anti-diabetic potential of 4 kinds of dark tea (Camellia sinensis L.) protein hydrolysates.《Journal of Applied Botany and Food Quality》.2019,第92卷57-63. * |
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